Adeno-associated virus (AAV), a non-pathogenic parvovirus, is an attractive vehicle for gene therapy. Serotypes of AAV exhibit both diversity in cellular tropism and long-term transgene expression, although its medical application is limited since infection is highly inefficient. AAV is an inefficient virus partially because cell surface attachment and endocytosis of the virus does not always lead to transgene expression, as successful infection depends on AAV escaping the endosome, trafficking correctly through the cytosol, and delivering its genome to the nucleus. Current research focuses on identifying functional domains of the viral capsid that permit efficient infection of specific cell types in order to advance the design of ideal virions for gene therapy. One of three capsid proteins, viral protein 1 (VP1), contains a unique part (VP1up) that possesses a putative phospholipase motif and nuclear localization signal, and is necessary for subcellular events of AAV infection. Though the function of VP1up is not entirely clear, similar domains in related parvoviruses are known to be more robust than those in AAV. Thus, the following aims are proposed: 1) Elucidate the mechanism of how VP1up modulates the endosomal escape, subcellular trafficking, and transgene delivery of AAV;2) Determine if AAV transduction can be enhanced when VP1up is mutated by rational design or random domain shuffling;and 3) Establish in vivo if tissue tropism is altered or infection efficiency is increased in the brain when an alternative VP1up is substituted into AAV. In short, these aims will be achieved by molecularly engineering chimeric AAV virions that incorporate altered VP1 proteins and then testing these virions for DNA packaging, cellular attachment, phospholipase activity, endosomal escape, nuclear targeting, and transgene expression using a variety of assays. In addition, virus infection will be imaged in cell lines in vitro by time-course immunofluorescence and in mouse brain in vivo by tracking bioluminescent virions. AAV is currently being tested as a gene delivery vehicle in several clinical trials and it is becoming increasingly important to maximize delivery into target tissues for specific and efficient therapy. This project will help to more clearly define the roles of viral components necessary for efficient AAV-mediated gene delivery in the brain. Through manipulating these components, it will soon be possible to design virions that are specialized for delivering therapeutics to cells affected by many different genetic disorders and diseases.